Assay of cholesterol 7α-hydroxylase activity in rat hepatocytes in primary monolayer culture

ANALYTICAL BIOCHEMISTRY 171, 158-165 (1988)

Assay of Cholesterol

7a-Hydroxylase Activity in Rat Hepatocytes Primary Monolayer Culture

in

HANS M. G. PRINCEN, PIET MEIJER, JAAP KWEKKEBOOM, AND HERMAN JAN M. KEMPEN Gaubius Institute TNO. Herenstraat 5d, 2313 AD Leiden, The Netherlands Received September 8, 1987 A sensitive and precise method is described to assay cholesterol 7a-hydroxylase activity in homogenates of rat hepatocytes cultured in monolayers for up to 76 h. The assay is based on measurement of the amount of radioactive cholesterol converted into 7a-[?I]hydroxycholesterol. Since no subcellular fractionation was applied to measure enzyme activity, this method is rapid and can be performed with cell protein, corresponding to as little as 1 to 2 million hepatocytes. Optimal assay conditions were determined and the reproducibility of this cholesterol ‘la-hydroxylase determination was established. Exogenous cholesterol ( 105 PM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent K,,, of 56 PM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7cY-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7a-hydroxylase activity in cultured hepatccytes revealed that after an initial loss of approximately 60% of the activity as compared with 287 pmol/h/mg for freshly isolated cells, the enzyme activity was increased to the initial level in hepatocytes cultured for 52 h. This result and the finding that the cholesterol 7a-hydroxylase activity was diminished by 94% after a 24-h incubation with 5 pM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis. Since the cholesterol 7cY-hydroxylase activity was a good reflection of the bile acid synthesis rate in hepatocytes during 76 h in culture, we conclude that this method may be a valuable tool in the study of the regulation of bile acid synthesis. 0 1988 Academic press, Inc. KEY WORDS: cholesterol; cholesterol 7a-hydroxylase; thin-layer chromatography; radioactivity measurement; cultured hepatocytes.

The liver plays an important role in the synthesis and catabolism of cholesterol (1). Conversion of cholesterol into bile acids takes place exclusively in the liver and represents the major pathway for elimination of cholesterol from the body. The microsomal cholesterol 7a-hydroxylase (EC 1.14.13.7) is the first and major rate-limiting enzyme in the bile acid synthesis pathway (l-4). The 7a-hydroxylase activity is believed to be regulated by the amount of bile acids returning to the liver via the portal vein and by nutritional and hormonal factors (3,4). Other studies revealed that cytosolic proteins (5-8) and phosphorylation-dephosphorylation (7,9,10) may be involved in the regulation of the cholesterol 7cy-hydroxylase activity. Most of the information concerning regulation of 0003-2697188 $3.00 Copyright 8 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

bile acid synthesis has been obtained from experiments using intact animals. In recent years, primary monolayer cultures of rat hepatocytes have been shown to be a valuable model for studying a variety of metabolic functions in the liver, including bile acid synthesis ( 1 l- 14). This in vitro system offers the opportunity to investigate the effects of mediators thought to be involved in cholesterol and bile acid metabolism (e.g., bile acids (15,16), lipoproteins (11,17), or hormones) directly on hepatocytes in a controlled and chemically defined environment. Moreover, the (side-)effects of several drugs, currently applied in clinical practice, on the synthesis of bile acids may conveniently be determined using monolayer cultures of hepatocytes (14,15). 158

ASSAY OF CHOLESTEROL

~wHYDROXYLASE

Most studies on the regulation of bile acid synthesis in hepatocyte monolayers, however, have been hampered by the difficulty in measuring the activity of the rate-limiting cholesterol 7a-hydroxylase in cultured cells. Although two reports (12,18) have been published, describing the measurement of enzyme activity in microsomes isolated from cultured rat hepatocytes, in these investigations large amounts of cells were needed (40-70 x 106) or hepatocytes were kept in suspension culture for only 5 h (18). In the present paper, we report a rapid and sensitive assay method for cholesterol 7cu-hydroxylase in homogenates of l-2 X lo6 rat hepatocytes, cultured for 76 h, using measurement of 14C radioactivity. In this work, the contribution of endogenous free cholesterol as substrate for the enzyme was also studied. MATERIALS

AND METHODS

Chemicals and animals. [4-‘4C]Cholesterol (60 mCi/mmol), [3H]acetic anhydride (50 mCi/mmol), and Enhance spray were purchased from New England Nuclear (Boston, MA). Culture medium and fetal bovine serum were from Flow Laboratories (Irvine, Scotland). Collagenase type I, soybean trypsin inhibitor, bovine serum albumin, dexamethasone, pyruvic acid, dithiothreitol (DTT)‘, butylated hydroxytoluene (BHT), and Tween 80 were obtained from Sigma Chemicals (St. Louis, MO). 7-Ketocholesterol (3&hydroxy-5-cholesten-7-one), 7aand 7@hydroxycholesterol, and 3/3,5a, 6P-trihydroxycholestane were from Steraloids (Wilton, NH). Glucose 6-phosphate, NADP, glucosed-phosphate dehydrogenase, NADH, and lactate dehydrogenase (from rabbit muscle) were purchased from Boehringer (Mannheim, FRG). Insulin (Actrapid, 40 III/ml) was from Novo Industri (Copenhagen, Denmark). Trypan blue was from ’ Abbreviations used: DTT, dithiothreitol; BHT (butylated hydroxytoluene), 2,6di-t-butyl-pcresol; Tween 80: polyoxyethylene sorbitan monooleate; Hepes, 4-(2hydroxyethyl)- 1-piperazineethanesulfonic acid.

IN CULTURED

HEPATOCYTES

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BDH Chemicals (Poole, Dorset, England). Cholesterol, Hepes, TLC precoated silica gel 60, and aluminium oxide (type E) plates (0.25 mm thickness) and all salts and solvents were obtained from E. Merck (Darmstadt, FRG). X-Omat AR films for autoradiography were purchased from EastmanKodak Company (Rochester, NY). Radioactive markers 7a- and 7@[4-‘4C]hydroxycholesterol were prepared from 7-[4“C]ketocholesterol, which is the most pronounced impurity in commercially available [4-‘4C]cholesterol, by reduction with sodium borohydride ( 19). 26-Hydroxycholesterol was a gift from Dr. N. B. Javitt, Division of Hepatic Diseases, New York University Medical Center (New York, NY). Male Wistar rats 250-350 g were used throughout and maintained on standard chow (Hope Farms, Woerden, The Netherlands) and water ad libitum. For preparation of hepatocytes animals were killed between 9 and 10 AM. Rat hepatocyte preparation and culture. Rat liver cells were isolated by perfusion with 0.05% collagenase and 0.005% trypsin inhibitor as described previously (20). Viability, as determined by trypan blue (0.11%) exclusion, was higher than 86%. The cells were seeded on 60-mm-diameter plastic tissue culture dishes (Costar, Cambridge, MA) at a density of 1 X lo5 cells/cm2 and were routinely maintained in Williams’ E medium supplemented with 10% heat-inactivated fetal bovine serum, 2 InM L-glutamine, 20 mu/ml insulin, 50 nM dexamethasone, 100 IU/ml penicillin, and 100 &ml streptomycin at 37°C in a 5% C02/95% air atmosphere. For each time point three to four dishes were plated. After a 4-h attachment period and every 24 h thereafter, medium was refreshed with 2.5 ml of culture medium. Hepatocytes were cultured for 3 days under these conditions and remained viable, as judged by trypan blue exclusion (more than 90% after 76 h) and leakage of the cytoplasmic enzyme lactate dehydrogenase to the culture medium. Lactate dehydrogenase activity (determined as described in Ref. (21)) in culture medium was less than 10% of cel-

160

PRINCEN ET AL.

lular content and decreased during culture time, in agreement with others (22). Cholesterol 7whydroxylase assay. Under Results and Discussion we will describe the various experiments leading to the selection of optimal conditions for the cholesterol 7~ hydroxylase assay. Immediately after isolation and cell count, a part of the hepatocyte suspension was centrifuged at 4°C for 5 min at 75g. Cells were resuspended and washed three times in cold Hanks’ buffer without Ca” and Mg*+, the cell pellet was then quickly frozen in liquid nitrogen in portions of 5- 10 X lo6 cells, and stored at -80°C routinely (t = 0 h sample). Cultured hepatocytes were harvested at indicated time points in the above buffer, washed three times, scraped using a rubber policeman, and frozen as described for t = 0 h samples. For determination of the cholesterol 7a-hydroxylase activity cell pellets were thawed rapidly and resuspended in 250 ~1 of cold hypotonic buffer containing 20 mM potassium phosphate, pH 7.4, 5 mM DTT, and 1 mM EDTA. Cells were disrupted at 4°C by 25 downward passes in a hand-driven 2-ml Potter-Elvehjem homogenizer equipped with a Teflon pestle. One milliliter of 100 mM potassium phosphate buffer, pH 7.4, plus 5 InM DTT was added and the homogenate was homogenized a further 15 strokes. Samples were taken for determination of protein and cholesterol, which were measured according to Lowry et al. (23) and Gamble et al. (24), respectively. An aliquot of the homogenate, containing l-2 mg of protein, was used to assay cholesterol 7a-hydroxylase activity. Homogenates in duplicates were preincubated for 10 min at 37°C in a medium containing 50 mM potassium phosphate, pH 7.4, 2.5 mM DTT, 105 PM [4-‘4C]cholesterol (0.3 &i, purified before use), and 1.5 mg/ml Tween 80. Enzyme activity at the end of the preincubation time was assayed by addition of a NADPH regenerating system containing 20 mM glucose 6-phosphate, 2 mM NADP, 4 mM MgC12, and 1.4 IU glucosed-phosphate dehydrogenase to a final volume of 1.0 ml.

After 30 min at 37 “C in a shaking water bath with exclusion of light the reaction was stopped by addition of 10 ml chloroform/ methanol 2: 1 containing 0.01% BHT. The organic layer was washed with 2 ml 0.9% NaCl and evaporated under nitrogen. The residue was applied to a thin layer of silica which was twice developed with toluene/ethylacetate 2:3, using 7a- and 7/3-[4-i4C]hydroxycholesterol as radioactive markers. Usually, plates were autoradiographed for 6 to 8 days at -80°C to visualize 7a-hydroxycholesterol and cholesterol; however, exposure times could be reduced to 2-3 days after spraying the plates with Enhance. For rapid determinations 30-50 pg 7~ and 7@-hydroxycholesterol and cholesterol were added as markers to the residue after evaporation and visualized with Rhodamine 6B. Spots containing 7a-hydroxycholesterol and cholesterol were scraped off and counted, using the [4-‘4C]cholesterol input as recovery standard. Blank values, determined by running parallel incubations without a NADPH generating system, were subtracted before calculating enzyme activity. Measurement of the mass of 7a-hydroxycholesterol formed was performed by the double isotope derivative procedure, as previously described by Mitropoulos and Balasubramaniam (25) and Shefer et al. (26), using the conditions of Shefer et al. Bile acid synthesis in cultured hepatocytes. Synthesis of bile acids in primary cultures of hepatocytes was determined by measuring conversion of 0.15 &i [4-‘4C]cholesterol per 10 cm* of cells into bile acids, accumulated during 24-h periods after 28, 52, or 76 h of incubation as previously reported ( 14). RESULTS AND DISCUSSION

Properties of the Enzyme System To achieve equilibration of exogenous with endogenous cholesterol, serving as a substrate for the enzyme, incubation mixtures were preincubated for various time intervals. Maximum enzyme activity was reached after a lo-min incubation, in agree-

ASSAY OF CHOLESTEROL

A j/l/i

7~HYDROXYLASE

IN CULTURED

HEPATOCYTES

161

formed for 30 min to assure optimal assay conditions. Under similar conditions using +NADPH rat liver microsomes, we observed that formation of 7a-hydroxycholesterol was linear for at least 90 min and up to 1.5 mg added -NACfH microsomal protein ( 14). Protein concentration (mglml) As depicted in Fig. 1C endogenous choles5w[ B terol in hepatocytes ( 10.2- 18.0 pg/mg, Table 3 2) was not sufficient to saturate the enzyme. In the presence of Tween 80, however, the enzyme appeared to be saturated at about 120 PM, a concentration well below the amount of cholesterol usually available to the enzyme in the standard assay (130- 170 PM). With the graphical method of Lineweaver-Burk an apparent K, for the enzyme of 56 PM was found. The difference between this value and the K,,, for cholesterol in an assay with microsomes (105 PM, Ref. (14)) may be explained by the presence of cytosolic activators in the homogenates fortifying FIG. 1. Effect of increasing amounts of homogenate the formation of 7cu-hydroxycholesterol (A), time (B), and substrate concentration (C) on choles(5-8). It has been suggested that one of these terol 7cy-hydroxylase activity in homogenates of hepatocytes after isolation (t = 0 h samples). Standard assay proteins, the cytosolic sterol carrier protein* conditions were employed, except for protein concenfacilitates introduction of substrate to the tration in (A), incubation time in (B), and substrate conmembrane-bound cholesterol 7a-hydroxycentration in (C). Protein in (B) and (C) was 1.66 mg/ml. lase (27,28). (A) Incubations in the presence (0) and absence (A) of Participation of endogenous cholesterol to NADPH regenerating system (blank). Values of incubathe enzymatic reaction was determined by tions without NADPH were corrected for nonspecific autoxidation as observed in incubation mixtures withquantitative acetylation of the assay mixture out homogenates (see Results and Discussion). Similar or the biosynthetic 7a-hydroxycholesterol, findings were observed with homogenates of hepatocytes separated on TLC after incubation, with cultured for various times. [3H]acetic anhydride to measure the mass of 7cu-hydroxycholesterol formed (25,26). Under the conditions applied, it was estiment with others (7). Using this preincubamated that approximately 70% of cholesterol tion time, optimal assay conditions were de- present in the homogenates was directly actermined. These are illustrated in Fig. 1. The cessible to the enzyme. This value is comparate of formation of 7a-hydroxycholesterol rable with the percentage of cholesterol in was linear with respect to total cell protein 9OOOg supernatants of rat liver available as concentration up to 2.5 mg/ml (2.5 mg per substrate for the enzyme (60-70%, Refs. assay, Fig. 1A). In this figure also the abso- (29,30)). For calculation of the amount of lute requirement for NADPH in the reaction cholesterol participating in the enzyme reacmedium is shown. The reaction rate was tion, we utilized the amount of endogenous proportional to the incubation time during cholesterol X 0.7 plus the amount of added the first 45 min after addition of the NADPH exogenous cholesterol, both in nanomoles. regenerating system (Fig. 1B). Therefore, in Blank values, obtained by running parallel standard incubations the amount of protein incubations without NADPH regenerating did not exceed 2 mg and reactions were per- system, were 60 to 70 pmol7cu-hydroxycho-

162

PRINCEN ET AL.

lesterol per 30-min incubation (under standard assay conditions). Similar values were observed after incubation of exogenous cholesterol without homogenate or after addition of boiled homogenates to reaction mixtures supplied with NADPH generating system. This observation indicates that 60 to 70 pmol of 7a-hydroxycholesterol was formed by nonspecific autoxidation of cholesterol and that no NADPH was present or biosynthesized in cell homogenates during the reaction. Subtraction of zero time blanks, as employed by several investigators, led to an overestimation of the reaction rate, since we found that 30 to 40 pmol of 7cY-hydroxycholesterol originated during the 40-min preincubation and incubation. The sensitivity of the method is such that 10 pmol of 7a-hydroxycholesterol can be detected. Cells were routinely cultured (14,32) in Williams’ E medium supplemented with 10% FCS and insulin and dexamethasone. These hormones greatly increase cell viability in primary monolayer cultures of rat hepatocytes (33,34). A representative autoradiograph of a TLC of extracts of 7a-hydroxylase assays with homogenates of cultured rat hepatocytes is shown in Fig. 2. By comparison of lanes containing metabolites of reactions carried out with or without NADPH, it can be seen that during incubation [4-14C]cholesterol was autoxidized (5,25,30) giving the following products: 3&S&,6/3-trihydroxycholestane, 7B-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol as identified by TLC, and several unidentified nonpolar compounds. In addition, a small amount of 7a-[4-‘4C]hydroxycholesterol was present in added [4-14C] cholesterol and formed nonspecifically during the 40-min preincubation and incubation (see remarks above). However, two metabolites were NADPH-dependently synthesized, 7a-hydroxycholesterol and a product running together with 25-hydroxycholesterol. This compound was identified using another TLC system (hexane/ethylacetate/aceton/2-propanol 85:15:2:3.5) and GLC-MS as 26-hy-

FIG.2. Autoradiographs of thin-layer chromatograms of the extracts from cholesterol 7cr-hydroxylase assays with homogenates of rat hepatocytes. Incubations were performed with homogenates of hepatocytes cultured for 0 h (lanes I and 2) 28 h (lanes 3 and 4), 52 h (lanes 5 and 6), and 76 h (lanes 7 and 8) with (+) or without (-) NADPH generating system (blanks). Enzyme activities in this experiment, in which one of a duplicate incubation is shown, were for t = 0 h 2 17 + 3 pmol/h/mg, for t = 28 h 94 f II pmol/h/mg, fort = 52 h 341 * 11 pmol/h/mg, and for t = 76 h 140 + 3 pmol/h/mg. The autoradiograph was exposed for 9 days without using Enhance spray. The positions of reference compounds (b-g) are indicated. a, start; b, 30,5d,6@-trihydroxycholestane; c, 7u-hydroxycholesterol; d, 7&hydroxycholesterol; e, 7-ketocholesterol; f, 25 and 26-hydroxycholesterol; and g, cholesterol.

droxycholesterol, thought to be formed by a mitochondrial 26-hydroxylase (1) in a NADPH-dependent manner. It is clear from this autoradiograph that no metabolites of 7a-hydroxycholesterol originate, as might be expected, since the next step in bile acid synthesis, the formation of 7Lu-hydroxy-4-cholesten-3-one, needs NAD+ as a cofactor ( 1,4). Furthermore, no radioactivity was detected in the methanol-water layer after extraction. Eflect of Storage, CO, and Potassium Fluoride on Enzyme Activity No cholesterol 7Lu-hydroxylase activity was lost during freezing and storage for 5 to 10 months, using the procedure described under Materials and Methods (Table 1). Usually enzyme activity was determined within 2 weeks after hepatocyte isolation. Incubation under a CO atmosphere completely inhibited the formation of 7a-hydroxycho-

ASSAY

OF

CHOLESTEROL

7a-HYDROXYLASE TABLE

IN CULTURED

HEPATOCYTES

163

1

CHOLESTEROL 7a-HYDROXYLASE AC-WITIES IN HEPATOCYTES ARER STORAGE AND AFTER INCUBATION UNDER CO AND WITH 50 mM KF Cholesterol 7a-hydroxylase activity (pmol/h/mg) Time of storage: Experiment Experiment Experiment Experiment Experiment +co

+50 mM

1b 2 3 4 4’ w

0 h”

1 week

152 f 13 296 f 4

143 f 14

2 months 164f 11 312 f 17

457 + 43 217+

3

228 f

5

152 201*

4 9

0 228 + 7

5 months

10 months

299 f 14 443*31 232 f 17

263 f 2’ 435 f 9

’ Cholesterol 7a-hydroxylase activities were determined immediately after hepatocyte isolation. Freezing in liquid nitrogen and rapid thawing (as described under Materials and Methods) did not change activities. b Experiments l-4 were performed with t = 0 h samples. Similar results were obtained in several experiments with hepatocytes cultured for 28, 52, and 76 h. ’ A representative experiment out of two (CO) or three (KF) different experiments is given. Values shown are means (*SD) of experiments with hepatocy-tes of one isolation with duplicate incubations. * Significant difference (P i 0.05) between freshly isolated cells and cells stored for the indicated period.

lesterol. In agreement with others (8,31) no indication was obtained about involvement of phosphorylation-dephosphorylation regulation for cholesterol 7a-hydroxylase (7,9,10). Enzyme activity did not differ upon homogenization and incubation in the presence or absence of 50 mM RF (Table 1). Cholesterol 7wHydroxylase Hepatocytes

Activity in Rat

Figure 2 also indicates that there is a difference in cholesterol 7a-hydroxylase activity in rat hepatocytes cultured for various times. Measured values of the specific rat hepatocyte culture shown in Fig. 2 and mean results of eight cultures are given in the legend of Fig. 2 and in Table 2, respectively. There was considerable variation in specific activity of the enzyme in hepatocytes from different animals, especially in t = 0 h samples. Similar variations have been reported by Hylemon et al. (12) in microsomes of rat hepatocytes prepared immediately after isolation and by us in rat liver microsomes (35). It is possible that this variation represents in-

terindividual differences, since no correlation was found between good and less optimal hepatocyte isolations (viability ranging from 86 to 95%). The existence of large interindividual variations in the rat of drug metabolism, particularly by the mixed-function oxidase system as a result of genetic factors, has been described (36,37). Since cholesterol 7a-hydroxylase is a cytochrome P-450-dependent enzyme (1,3,4) a similar phenomenon may occur with this enzyme. Hepatocytes lost cholesterol 7a-hydroxylase activity during culturing for 28 h (63%). However, enzyme activity was restored to initial levels after 52 h and decreased afterward. Loss of total cytochrome P-450 has been well established as one of the phenotypic changes associated with adaptation of hepatocytes to conditions of monolayer culture (for a review, see Ref. (38)), but there may be marked heterogeneity in the rates at which individual P-450 forms are lost in culture (39). Here we report that, after an initial loss, there is again an increase of this specific cytochrome P-450 activity in primary monolayer cultures. A similar pattern has been

164

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ET AL.

TABLE

2

CHOLESTEROL 7a-HYDROXYLASE ACTIVITIES, CELLULAR CHOLESTEROL CONCENTRATIONS, AND BILE ACID SYNTHESISIN CULTURED RAT HEPATOCYTES Age of culture

Cholesterol 7a-hydroxylase

h

n

pmoVh/mg

0 28 52 76

10 8 8 8

287 f 105 + 250 + 102+

153 46 67 46

52 52

2 3

69 + 15+

16 4

Free cholesterol

Soft=0

Wmg)

n

Bile acid synthesis @pm/24 Wmg)

9 9 9

4870 rt 1390 8690 f 2010 6250 f 1800

Control

Cycloheximide 1 PM 5pM

37 87 36

10.2 13.4 17.1 18.0

f + f +

1.3 1.8 1.7 3.6

15.7 + 2.9 13.4 + 1.4

Note. Cholesterol 7d-hydroxylase activities and cellular free cholesterol concentrations were determined in cells harvested at the indicated times. Bile acid synthesis was measured by determination of radioactivity in bile acids, accumulated in cells and medium during the 24-h period preceding the indicated time. Cycloheximide was present during a 24-h period preceding the mentioned time. Values shown are means (*SD) of experiments with hepatocytes of n different rats with duplicate incubations and are expressed per milligram of total cell protein. Cholesterol 7a-hydroxylase activity in t = 0 h samples ranged between 153 f 6 and 592 & 5 1 pmol/h/mg.

found by Hylemon et al. (12). This observation, together with the fact that after addition of 5 pM cycloheximide to the culture medium the 7a-hydroxylase activity dramatically decreased (Table 2), suggests that cholesterol 7cu-hydroxylase activity results from de nova protein synthesis during the 76-h culture period. Cholesterol 7+hydroxylase activity perfectly paralelled bile acid synthesis as measured by conversion of [4-i4C]cholesterol into bile acids (Table 2). With respect to this, it should be realized, that measurement of enzyme activity gives an instantaneous reflection of bile acid synthesis rate, whereas determination of bile acids formed over a 24-h period is a cumulative process. In conclusion, we have described an accurate and sensitive method to measure cholesterol 7cu-hydroxylase activity in hepatocytes cultured for up to 76 h. In contrast with previously described methods ( 12,18), the procedure is rapid and can be performed with a small amount of cells ( 1 to 2 X 10% since

enzyme activity is determined directly in homogenates. Our results indicate that measurement of cholesterol 7a-hydroxylase in primary monolayers of cultured hepatocytes is a reliable method and, in addition to measuring bile acid biosynthetic rates, is a useful extension to the study of regulation of bile acid synthesis. ACKNOWLEDGMENTS The authors thank Mr. H. van der Voort for performing lipid analysis and Mrs. C. Horsting-Been and Miss M. Horsting for typing the manuscript. Dr. E. van Heett is gratefully acknowledged for performing GLC-MS determinations.

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ASSAY OF CHOLESTEROL

7c~-HYDROXYLASE

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